The IP3 receptor is the major ion channel which controls the release of Ca2+ into the cytosol from the endoplasmic reticulum. IP3R mediated Ca2+ signals are involved in a large number of cellular physiological processes, including maintaining normal cell bioenergetics, gene expression, and learning and memory. IP3Rs can regulate a large number of cellular processes due to their ability to generate an enormous range of spatiotemporally patterned Ca2+ signals. IP3Rs open in response to elevated concentrations of IP3, but have an interesting property - they are also biphasically gated by the concentration of Ca2+, such that very high or very low concentrations of Ca2+ decrease the open probability, while moderate concentrations cause them to open. Ca2+ can flux out of an open channel and diffuse towards another, triggering the neighboring channel to open in a feedforward reaction termed calcium induced calcium release (CICR). The strength of communication between IP3Rs is therefore determined by the spacing between them. Although immunohistochemistry shows that IP3Rs are evenly spread throughout the ER, only small clusters of IP3Rs give rise to Ca2+ release events, called 'puffs', which repeatedly arise from the same locations. The properties distinguishing these functionally active IP3Rs from inactive IP3Rs remain completely undetermined. In a previous study, overexpression of fluorescently labeled IP3Rs revealed a subpopulation of immotile receptors. It remains to be shown if the immotile population of IP3Rs and the IP3Rs which give rise to puffs are one and the same. The focus of this research proposal is to determine the nanoarchitecture of a cluster of functional IP3Rs using Ca2+ imaging (Aim 1) and to determine the distribution and motility of functionally active IP3R proteins by combining superresolution microscopy and Ca2+ imaging (Aim 2). Findings from this proposal will provide mechanistic insight into the generation and propagation of Ca2+ signals, which play a critical role in many basic biological processes.